1. Field of the Invention
The present invention is related to the copying or amplification of nucleic acid molecules in the presence of gellan gum.
2. Description of the Related Art
The PCR method of DNA amplification is widely used in research, forensic investigations, and medical diagnostics. PCR will amplify even a few molecules of a target DNA in a sample to the level of 107 to 108 molecules within a short period (1-2 hours). PCR is one of a few technologies that will detect or amplify relatively few target nucleic acid molecules. There is great interest in enhancing this ability of PCR reactions to amplify a few target nucleic acid sequences, or its “sensitivity”. Many such techniques to optimize PCR reactions are described, for example, in Section 15, Vol. 3, “Current Protocols in Molecular Biology” (Edit. Ausubel, F. M., et al), John Wiley & Sons).
For several applications of nucleic acid amplification, it is desirable to carry out the amplification within, or associated with, a gel matrix. Many types of gel material suitable for use as electrophoresis medium have been the subject of intense research as the gel is often the determining factor for a successful separation. The gels may be composed of natural materials, e.g., agarose or synthetic polymers, e.g., polyacrylamide.
When the PCR ingredients are polymerized within a polyacrylamide gel, the resulting PCR amplifications are inconsistent, perhaps due to damage to enzymes and/or nucleic acids by the free radicals necessary to polymerize the acylamide (BioTechniques 33(1), 150-156, 2002; Chetverina, H. V. et al. “Molecular Colony Diagnostics: detection and quantification of viral nucleic acids by in-gel PCR.”; and Nucleic Acid Research, 27(24), e34, 1999; R. D. Mitra and G. M. Church. “In situ localized amplification and contact replication of many individual DNA molecules.”).
Agarose inhibits PCR amplification when the agarose concentration is above 0.15%. (Biotechiques 33(2), 282-283, 2002. Yamaguchi, Y, et al “Inhibitory effects of Agarose Gel and LB medium on DNA sequencing.”). Agar contains uncharacterized inhibitory components that block PCR even when present in only trace amounts in DNA purified from cells growing on the surface of agar plates. (Journal of Clinical Microbiology 36, 275-276, 1998 “Inhibition of PCR by agar from bacteriological transport media”).
Therefore, there exists a need in the art for methods of enhanced PCR sensitivity so as to be able to amplify from samples that contain low levels of nucleic acid molecules.